Multiple Assay Device

ABSTRACT

Disclosed is an assay device for the determination of analyte in a liquid sample over an extended concentration range comprising a first assay and a second assay, wherein the first assay for an analyte comprises a first flow-path having a sole detection zone capable of immobilising a labelled binding reagent and the second assay for said analyte comprises a second flow-path having a sole detection zone capable of immobilising a labelled binding reagent, wherein the presence of labelled binding reagent at the detection zones provides an indication of the presence and/or extent of analyte in said liquid sample.

RELATED APPLICATIONS

The present application claims benefit of priority to U.S. ProvisionalPatent Application No. 60/991,531, filed on Nov. 30, 2007; Great BritainApplication 0706906.5, filed Apr. 10, 2007; and Great BritainApplication 0717043.4, filed Sep. 1, 2007, the contents of which areincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to an assay device, kit and method fordetermining the presence or concentration of an analyte over an extendedconcentration range.

BACKGROUND OF THE INVENTION

Simple lateral flow immunoassay devices have been developed andcommercialised for detection of analytes in fluid samples, see forexample EP291194. Such devices typically comprise a porous carriercomprising a dried mobilisable labelled binding reagent capable ofbinding to the analyte in question, and an immobilised binding reagentalso capable of binding to the analyte provided at a detection zonedownstream from the labelled binding reagent. Detection of theimmobilised labelled binding at the detection zone provides anindication of the presence of analyte in the sample.

Alternatively, when the analyte of interest is a hapten, the immunoassaydevice may employ a competition reaction wherein a labelled analyte oranalyte analogue competes with analyte present in the sample for animmobilised binding reagent at a detection zone. Alternatively the assaydevice may employ an inhibition reaction whereby an immobilised analyteor analyte analogue is provided a detection zone, the assay devicecomprising a mobilisable labelled binding reagent for the analyte.

A sandwich immunoassay is often the assay of choice when detectinganalytes. However, a sandwich assay is not always possible, for examplein the case of small molecules such as haptens which may not be largeenough to allow the simultaneous binding thereto of two differentbinding partners. A dose-response curve prepared using a typical lateralflow device employing a sandwich immunoassay shows increasing levels ofsignal with increasing analyte up to the point where at higher analytelevels the curve tends to plateau. At yet higher analyte levels, thesignal begins to decrease due to preferential capture at the detectionzone of analyte which has not yet bound to labelled reagent. Thisphenomenon is known as the hook effect. Thus sandwich immunoassaysexhibit a limited assay range due to the fact that the signal amount orintensity observed at higher analyte levels may be the same, or evenless, than that observed at lower analyte levels.

A competition or inhibition assay typically provides a high signal atzero or low levels of analyte. At increasing levels of analyte thesignal level may still be high depending upon the amount of labelledbinding species present compared to the amount of analyte. At stillincreasing levels of analyte, the signal starts to decrease as unboundanalyte either competes with labelled analyte or analyte analogue forthe immobilised binding reagent or binds to labelled binding reagent,lowering binding of the unlabelled binding reagent at the detectionzone.

Thus the above assay methods are not suitable for measuring levels ofanalyte over an extended analyte range.

US2005/0112780 discloses an assay device and method for extending thedynamic detection range of assay devices comprising a flow throughporous carrier comprising a detection zone and a compensation zoneprovided downstream from the detection zone. The detection involves afirst binding reagent which binds a detection probe to generate adetection signal having an intensity proportional to the amount ofanalyte, and the compensation zone comprises a second capture reagentwhich binds a detection probe to generate a signal which is inverselyproportional to the intensity of the detection signal. The assay mayfurther comprise a third calibration zone which generates a signal. Thefirst binding reagent may be selected from a group including an antigen,hapten or streptavidin. The first and second binding reagents may bechosen from a number of species including an antigen, hapten orstreptavidin.

US2004/0197820 discloses a flow through porous carrier assay device forreducing the hook effect comprising a detection zone wherein the devicemay include a downstream calibration zone.

US2006/0019404 discloses an assay device with an extended dynamic rangecomprising a lateral flow test-strip comprising a plurality of detectionzones with a progressively decreased sensitivity to analyteconcentration. The assay device may comprise two carriers each having aplurality of detection zones. The amount of label/signal present at theplurality of detection zones is detected to determine the analyteconcentration.

EP462376 discloses an assay device comprising a capture site and aconjugate recovery site wherein the conjugate recovery site receives andbinds said conjugate or conjugate complexes which migrate through saidcapture site and wherein immobilised conjugate at both the conjugaterecovery site and capture site is detected to determine the amount ofthe analyte of interest.

The present inventors have shown that for assay devices wherein multipledetection zones for the detection of an analyte are provided on the sameporous carrier, binding at an upstream detection zone may change thebinding characteristics at a downstream detection zone and that anyvariation in binding at an upstream detection zone may cause acompounded variation of binding at a downstream detection zone. This isespecially so at higher analyte concentration levels and can give riseto poor assay precision. Furthermore, it has been found thatcross-binding may occur between the respective binding reagents presentin the detection zones during running of the test and cross-binding hasalso been observed during manufacture of the devices and whilst they arestored in the dry state. This was shown to have an impact on the levelsof assay precision and sensitivity. These problems do not appear to havebeen recognised previously in the prior art.

It is an object to provide an improved assay device, kit and method forextending the analyte range of an assay.

SUMMARY OF THE INVENTION

An assay device for determining the presence or concentration of ananalyte such as hCG in a liquid sample over an extended concentrationrange is provided. In one embodiment, the device comprises a firstflow-path having a sole detection zone capable of immobilising alabelled binding reagent and a second flow-path having a sole detectionzone capable of immobilising a labelled binding reagent, wherein thepresence of labelled binding reagent at the detection zones provides anindication of the presence or concentration of analyte in said liquidsample.

In another embodiment, an assay device may comprise a first flow-pathand a second flow-path wherein said first flow-path comprises a porouscarrier having a detection zone comprising an immobilised bindingreagent for an analyte and provided upstream from the detection zone isa mobilisable labelled binding reagent for said analyte; and whereinsaid second flow-path comprises a detection zone comprising animmobilised binding reagent for the analyte and a mobilisable labelledbinding reagent upstream from the detection zone for the analyte and ascavenger reagent for said analyte, wherein detection of the presence oflabelled binding reagent at the detection zone provides an indication ofthe presence or concentration of analyte in said liquid sample.

In another aspect, the first flow-path is capable of providing anindication of the level of analyte in a first concentration range andthe second flow-path is capable of providing an indication of the levelof analyte in a second concentration range. The assay device accordingis capable of providing an indication of the level of analyte withrespect to one or more thresholds. The first flow-path of the device maydefine a sandwich assay, and the second flow-path may define acompetition or inhibition assay.

The first flow-path may comprise a mobilisable labelled binding reagentfor the analyte provided upstream from a detection zone, wherein thedetection zone comprises an immobilised binding reagent for the analyteand wherein the second flow-path comprises a mobilisable binding reagentfor the analyte provided upstream from provided upstream from adetection zone, said detection zone comprising an immobilised analyte oranalyte analogue for the mobilisable binding reagent. The firstflow-path may define a high analyte sensitivity assay, and the secondflow-path defines a low analyte sensitivity assay.

Assay kits and methods for determining the presence or concentration ofan analyte in a liquid sample comprising an assay device comprising thedevices are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention are further illustrated by reference to thefollowing figures:

FIG. 1 illustrates typical signal responses that are observed for atypical assay compared with that of a typical competition assay.

FIG. 2 illustrates plots of signal intensity vs hCG concentration forExample 1 and Comparative Example 1.

FIG. 3 illustrates a plot of signal intensity vs hCG concentration forthe assay device according to Example 2.

FIG. 4 illustrates the effect of varying the amounts of scavengerantibody for the assay device according to Example 2.

FIG. 5 illustrates the effect of varying latex spray positions to reducea hook effect using a device as described herein.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise above, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Where a term isprovided in the singular, the inventor also contemplates the plural ofthat term. The nomenclature used herein and the procedures describedbelow are those well known and commonly employed in the art.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

As used herein, the term “analyte” generally refers to a substance to bedetected. The term “analyte” includes, but is not limited to, toxins,organic compounds, proteins, peptides, microorganisms, bacteria,viruses, amino acids, nucleic acids, carbohydrates, hormones, steroids,vitamins, drugs (including those administered for therapeutic purposesas well as those administered for illicit purposes), pollutants,pesticides, and metabolites of or antibodies to any of the abovesubstances. The term “analyte” also includes any antigenic substances,haptens, antibodies, macromolecules, and combinations thereof. Specificexamples of analytes include but are not limited to ferritin; creatininekinase MB (CK-MB); digoxin; phenytoin; phenobarbitol; carbamazepine;vancomycin; gentamycin; theophylline; valproic acid; quinidine;luteinizing hormone (LH); follicle stimulating hormone (FSH); estradiol,progesterone; C-reactive protein; lipocalins; IgE antibodies; cytokines;vitamin B2 micro-globulin; glycated hemoglobin; cortisol; digitoxin;N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, suchas rubella-IgG and rubella IgM; antibodies to toxoplasmosis, such astoxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM);testosterone; salicylates; acetaminophen; hepatitis B virus surfaceantigen (HBsAg); antibodies to hepatitis B core antigen, such asanti-hepatitis B core antigen IgG and IgM (Anti-HBC); human immunedeficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1and 2 (HTLV); hepatitis Be antigen (HBeAg); antibodies to hepatitis Beantigen (Anti-HBe); influenza virus; thyroid stimulating hormone (TSH);thyroxine (T4); total triiodothyronine (Total T3); free triiodothyronine(Free T3); carcinoembryoic antigen (CEA); lipoproteins, cholesterol, andtriglycerides; and alpha fetoprotein (AFP). Drugs of abuse andcontrolled substances include, but are not intended to be limited to,amphetamine; methamphetamine; barbiturates, such as amobarbital,secobarbital, pentobarbital, phenobarbital, and barbital;benzodiazepines, such as librium and valium; cannabinoids, such ashashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates,such as heroin, morphine, codeine, hydromorphone, hydrocodone,methadone, oxycodone, oxymorphone and opium; phencyclidine; andpropoxyhene.

“Antigen” shall mean any compound capable of binding to an antibody, oragainst which antibodies can be raised.

“Antibody” shall mean an immunoglobulin having an area on its surface orin a cavity that specifically binds to and is thereby defined ascomplementary with a particular spatial and polar organization ofanother molecule. The antibody can be polyclonal or monoclonal.Antibodies may include a complete immunoglobulin or fragments thereof,which immunoglobulins include the various classes and isotypes, such asIgA (IgA1 and IgA2), IgD, IgE, IgM, and IgG (IgG1, IgG2, IgG3, and IgG4)etc. Fragments thereof may include Fab, Fv and F(ab′)₂, Fab′, and thelike. Antibodies may also include chimeric antibodies made byrecombinant methods.

A “binding reagent” refers to a member of a binding pair, i.e., twodifferent molecules wherein one of the molecules specifically binds withthe second molecule through chemical or physical means. The twomolecules are related in the sense that their binding with each other issuch that they are capable of distinguishing their binding partner fromother assay constituents having similar characteristics. The members ofthe specific binding pair (“sbp”) are referred to as ligand and receptor(antiligand), sbp member and sbp partner, and the like. In addition toantigen and antibody specific binding pair members, other specificbinding pairs include biotin and avidin, carbohydrates and lectins,complementary nucleotide sequences, complementary peptide sequences,effector and receptor molecules, enzyme cofactors and enzymes, enzymeinhibitors and enzymes, a peptide sequence and an antibody specific forthe sequence or the entire protein, polymeric acids and bases, dyes andprotein binders, peptides and specific protein binders (e.g.,ribonuclease, S-peptide and ribonuclease S-protein), and the like.Furthermore, specific binding pairs can include members that areanalogues of the original specific binding member, for example ananalyte-analogue or a specific binding member made by recombinanttechniques or molecular engineering. A molecule may also be a bindingpair member for an aggregation of molecules; for example an antibodyraised against an immune complex of a second antibody and itscorresponding antigen may be considered to be a binding pair member forthe immune complex. In addition to antigen and antibody binding pairmembers, other binding pairs include, as examples without limitation,biotin and avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence and an antibody specific for the sequence or theentire protein, polymeric acids and bases, dyes and protein binders,peptides and specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), and the like. Furthermore, specific bindingpairs can include members that are analogues of the original specificbinding member.

The terms “comprise” and “comprising” is used in the inclusive, opensense, meaning that additional elements may be included.

The term “flow-path” for the purposes of this invention refers to asubstrate that is able to convey a liquid from a first position to asecond position and may be for example a capillary channel, amicrofluidic pathway, or a porous carrier such as a lateral flow porouscarrier. The porous carrier may comprise one or a plurality of porouscarrier materials which may overlap in a linear or stacked arrangementor which are fluidically connected. The porous carrier materials may bethe same or different. A first flow path of a device provides for afirst assay, and a second flow path of the device provides for a secondassay.

“Label” when used in the context of a labelled binding reagent, refersto any substance which is capable of producing a signal that isdetectable by visual or instrumental means. Various labels suitable foruse in the present invention include labels which produce signalsthrough either chemical or physical means, such as being opticallydetectable. Such labels include enzymes and substrates, chromogens,catalysts, fluorescent compounds, chemiluminescent compounds,electroactive species, dye molecules, radioactive labels and particlelabels. The analyte itself may be inherently capable of producing adetectable signal. The label may be covalently attached to the bindingreagent.

“Labelled binding reagent” refers to any substance comprising adetectable label attached to a binding reagent. The attachment may becovalent or non-covalent. The label provides a detectable signal that isrelated to the presence or amount of analyte in the fluid sample.Various labels suitable for use include labels which produce signalsthrough either chemical, biochemical or physical means. Such labels caninclude enzymes and substrates, chromogens, catalysts, fluorescentcompounds, chemiluminescent compounds, and radioactive labels. Othersuitable labels include colloidal metallic particles such as gold orsilver, colloidal non-metallic particles such as selenium or tellurium,dyed or colored particles such as a coloured polymer such aspolystyrene, a stained microorganism or dyesols, organic polymer latexparticles and liposomes, colored beads or electrochemically detectablespecies. Of the above, colored polymer particles and colloidal gold arepreferred.

The term “non-labelled binding reagent” for the purposes of thisinvention refers to a binding reagent that is either not labelled with adetectable label or is labelled with a label which is not detected inorder to determine the analyte.

“Porous carrier” refers to a porous body capable of transporting fluidsample.

The term “sample” refers to any sample potentially containing ananalyte. For example, a sample may be a bodily fluid such as blood,urine, mucous or saliva, or a respiratory sample, such as anasopharyngeal wash or aspirate, nasal swab, nasopharyngeal swab, nasalwash, throat swab, transtracheal aspirate, bronchoalveolar lavage,elution buffer used to wash a respiratory sample, etc.

For the purpose of this application the term “scavenger binding reagent”denotes an additional binding reagent capable of binding analyte and theterm “scavenger” is used merely to distinguish the binding reagents fromthe other binding reagents present in the device.

According to a first aspect, the invention provides an assay device forthe determination of analyte in a liquid sample over an extendedconcentration range comprising a first assay and a second assay, whereinthe first assay for an analyte comprises a first flow-path having a soledetection zone capable of immobilising a labelled binding reagent andthe second assay for said analyte comprises a second flow-path having asole detection zone capable of immobilising a labelled binding reagent,wherein the presence of the labelled binding reagent at the detectionzones provides an indication of the presence and/or concentration ofanalyte in said liquid sample.

The first assay may provide an indication of the level of analyte in afirst concentration range and the second assay may provide an indicationof the level of analyte in a second concentration range.

The first and second concentration ranges differ from each other. Thefirst and second concentration ranges may overlap so as to provide acontinuous concentration range.

The assay device may be capable of providing an indication of the levelof analyte with respect to one or more thresholds. The assay device mayprovide an indication of the level of analyte below or above a pluralityof thresholds. For example the number of thresholds may be two three,four, five or greater.

The first and second assays may either independently or together providean indication of the level of analyte within a certain range.

According to an embodiment, the first assay provides an indication ofthe level of analyte of less than or equal to a first threshold and thesecond assay provides an indication of the level of analyte of above orequal to a second threshold. The first and second assays togetherprovide an indication of the level of analyte of greater than the firstthreshold but less than the second threshold.

A non-labelled binding reagent for the analyte or an analyte analoguemay be provided in an immobilised form at a detection zone. Thenon-labelled binding reagent may be chosen from a binding reagent forthe analyte of interest, an analyte or analyte analogue, depending uponwhether the assay is a sandwich type assay or a competition type assay.Similarly the labelled binding reagent may comprise a labelled bindingreagent for the analyte of interest, a labelled analyte or labelledanalyte analogue.

Alternatively a reagent may be provided in an immobilised form at thedetection zone that is capable of binding a labelled bindingreagent-analyte-non-labelled binding reagent complex. For example thebinding reagent may be conjugated or otherwise joined to binding speciessuch as biotin, the reagent immobilised at the detection zone being acomplementary binding partner such as streptavidin.

The non-labelled binding reagent may be provided in a mobilisable formwhich is capable of immobilising a labelled binding reagent-analytecomplex at a detection zone. For example the non-labelled bindingreagent may be attached to particle such as agarose and the detectionzone may comprise a filter of dimensions smaller than the particle, butlarger than the size of the labelled binding reagent, such that thefilter is able to trap the any labelled bindingreagent-analyte-non-labelled binding reagent complex present, anylabelled binding reagent that is not complexed to the capture reagentbeing able to pass through the filter.

The first and/or second assay may comprise a labelled binding reagentprovided in a mobilisable form upstream from the detection zone reagentin the dry state prior to use of the device.

The first and second assays may each comprise a mobilisable labelledbinding reagent provided upstream from an immobilised non-labelledbinding provided at each detection zone.

The assay device may comprise more than two assays, each capable ofdetecting the analyte at a particular concentration range or above orbelow one or more thresholds.

The first and second assays may individually or together provide anindication of the particular level of analyte, or whether the analyte isabove or below a certain threshold.

The assay device may have a common sample application region thatfluidically connects the plurality of flow-paths. Thus a fluid sampleapplied to sample application region of the device is able to travelalong the flow-paths of the respective assays to the respectivedetection zones. The sample application region may comprise a poroussample receiver. In the case where the mobilisable labelled bindingreagent is the same for both assays it may be provided at the sampleapplication region.

As an alternative to providing the first and second assays within asingle assay device, the assays may be provided as separate assaydevices, the results from the respective devices when taken togetherbeing capable of providing an indication or measurement of the level ofanalyte.

Thus according to a second aspect, the invention provides an assay kitfor the determination of an analyte over an extended concentration rangecomprising a first assay device and a second assay device, wherein thefirst and second assay devices comprise respectively first and secondassays according to the first aspect of the invention.

According to a third aspect, the invention provides a method for thedetermination of an analyte over an extended analyte range comprisingthe steps of:

-   -   a) adding a liquid sample to a first assay comprising a        mobilisable labelled binding reagent provided upstream from a        sole detection zone and to a second assay comprising a        mobilisable labelled binding reagent provided upstream from a        sole detection zone, said detection zones being capable of        immobilising labelled binding reagent, and wherein detection of        labelled reagent at the detection zones provides an indication        of the concentration and/or presence of an analyte in the liquid        sample.    -   b) reading the result of the assay.

In the case where the level of analyte is known to vary as a function oftime, for example the pregnancy hormone hCG, the assay device mayprovide a time-based indication to the user, such as the duration ofpregnancy in units of days or weeks.

The first and second flow paths may be provided on separate substratesor they may be provided on a common substrate such that liquid beingconveyed along a flow-path of the first assay is not able to cross overto the flow-path of the second assay. For example, the first and secondassays may be provided on the same porous carrier such that the firstand second flow-paths are isolated from each other. This may be achievedfor example by laser cutting parts of the porous carrier to make itnon-porous, thus separating the first and second flow-paths. As yet afurther alternative, the first and second detection zones may beprovided on the same flow-path in substantially a side by sidearrangement, such that neither is provided downstream from the other.

In particular the flow-path may be a lateral flow porous carrier.Suitable materials that may be employed as a porous carrier includenitrocellulose, acetate fibre, cellulose or cellulose derivatives,polyester, polyolefin or glass fibre. The porous carrier may comprisenitrocellulose. This has the advantage that a binding reagent can beimmobilised firmly without prior chemical treatment. If the porous solidphase material comprises paper, for example, the immobilisation of theantibody in the second zone needs to be performed by chemical couplingusing, for example, CNBr, carbonyldiimidazole, or tresyl chloride.

The label may comprise a particle such as gold, silver, colloidalnon-metallic particles such as selenium or tellurium, dyed or colouredparticles such as a polymer particle incorporating a dye, or a dye sol.The dye may be of any suitable colour, for example blue. The dye may befluorescent. Dye sols may be prepared from commercially-availablehydrophobic dyestuffs such as Foron Blue SRP (Sandoz) and Resolin BlueBBLS (Bayer). Suitable polymer labels may be chosen from a range ofsynthetic polymers, such as polystyrene, polyvinyltoluene,polystyrene-acrylic acid and polyacrolein. The monomers used arenormally water-insuluble, and are emulsified in aqueous surfactant sothat monomer mycelles are formed, which are then induced to polymeriseby the addition of initiator to the emulsion. Substantially sphericalpolymer particles are produced. An ideal size range for such polymerparticles is from about 0.05 to about 0.5 μm. According to an exemplaryembodiment the label is a blue polymeric particle.

The liquid sample can be derived from any source, such as an industrial,environmental, agricultural, or biological source. The sample may bederived from or consist of a physiological source including blood,serum, plasma, interstitial fluid, saliva, sputum, ocular lens liquid,sweat, urine, milk, ascots liquid, mucous, synovial liquid, peritonealliquid, transdermalexudates, pharyngeal exudates, bronchoalveolarlavage, tracheal aspirations, cerebrospinal liquid, semen, cervicalmucus, vaginal or urethral secretions and amniotic liquid. In particularthe source is human and in particular the sample is urine.

A particular analyte is human chorionic gonadotropin (hCG). The analytemay have a sole binding region or epitope or may have more than onebinding region. For example the analyte hCG comprises an alpha sub-unitidentical to that of luteinising hormone (LH), follicle stimulatinghormone (FSH) and thyroid stimulating hormone (TSH) and a beta sub-unitunique to hCG. Antibodies to the alpha and beta sub-units may be used tobind to hCG in a sandwich immunoassay format.

The assay device of the invention may be used to measure the presence ofhCG or concentration over an extended range. The range may vary frombetween, for example, about 10 mIU to about 250,000 mIU, from betweenabout 30 mIU to about 200,000 mIU, from between about 50 mIU to about175,000 mIU, from between about 100 mIU to about 100,000 mIU, frombetween about 250 mIU to about 75,000 mIU, from between about 300 mlU toabout 50,000 mIU, or from between about 500 mIU to about 25,000 mIU.

According to an embodiment, the device is able to measure the amount ofhCG in the fluid sample and to indicate to the user, based upon storedreference values the duration of pregnancy in time based units. Thedevice may also indicate whether the subject is pregnant or not,determined by whether the level of hCG is above or below a basethreshold. The reference and threshold values are typically storedwithin the device as part of an algorithm. The base threshold maytypically range from 10-25 mIU/ml. The base threshold may be, forexample, 10, 15, 20 or 25 mIU/ml.

According to an embodiment, the first assay may provide either anindication of whether the subject is pregnant or not, based upon whetherthe level of hCG detected is respectively above or below a basethreshold, and/or if pregnant, the level of hCG in a first range of lessthan or equal to a first threshold, the second assay provides anindication of the level of hCG in a second range of above or equal to asecond threshold and wherein the first and second assays togetherprovide an indication of the level of hCG in a third range of greaterthan the first threshold but less than the second threshold.

The first and/or second assay may further comprise a control zone toindicate that the reagents have mobilised and have been transportedalong the flow path and that the assay test has been carried outsatisfactorily. The control zone is typically positioned downstream fromthe detection zone and may for example comprise an immobilised bindingreagent for a labelled binding reagent. The labelled binding reagent maybe present in a mobilisable form upstream from the control zone anddetection zone. The labelled binding reagent may the same or differentto the labelled binding reagent for the analyte.

The assay device may comprise a porous sample receiver in fluidconnection with and upstream from the first and second flow-paths. Theporous sample receiver may be provided within the housing or may atleast partially extend out of said housing and may serve for example tocollect a urine stream. The porous sample receiver may act as a fluidreservoir. The porous sample receiving member can be made from anybibulous, porous or fibrous material capable of absorbing liquidrapidly. The porosity of the material can be unidirectional (ie withpores or fibres running wholly or predominantly parallel to an axis ofthe member) or multidirectional (omnidirectional, so that the member hasan amorphous sponge-like structure). Porous plastics material, such aspolypropylene, polyethylene (preferably of very high molecular weight),polyvinylidene flouride, ethylene vinylacetate, acrylonitrile andpolytetrafluoro-ethylene can be used. Other suitable materials includeglass-fibre.

If desired, an absorbant “sink” can be provided at the distal end of thecarrier material. The absorbent sink may comprise of, for example,Whatman 3MM chromatography paper, and should provide sufficientabsorptive capacity to allow any unbound labelled binding reagent towash out of the detection zone. As an alternative to such a sink it canbe sufficient to have a length of porous solid phase material whichextends beyond the detection zone.

Following the application of a binding reagent to a detection zone, theremainder of the porous solid phase material may be treated to block anyremaining binding sites. Blocking can be achieved by treatment forexample with protein (e.g. bovine serum albumin or milk protein), orwith polyvinylalcohol or ethanolamine, or combinations thereof. Toassist the free mobility of the labelled binding reagent when the porouscarrier is moistened with the sample, the porous carrier may furthercomprise a material such as sucrose or lactose. Such material may bedepositing for example as an aqueous solution in the region to which thelabelled binding reagent is to be applied. Alternatively such materialmay be deposited upstream from or at the labelled binding reagent.

The nitrocellulose porous carrier may have having a pore size of atleast about 1 micron, for example greater than about 5 microns, and forexample about 8-12 microns.

The nitrocellulose porous carrier may be backed e.g. with a plasticssheet, to increase its handling strength. This can be manufacturedeasily by forming a thin layer of nitrocellulose on a sheet of backingmaterial such as Mylar™.

The labelled binding reagent may be provided on a separate macroporouscarrier material to that of the detection zone. The macroporous carriermaterial should be low or non-protein-binding, or should be easilyblockable by means of reagents such as BSA or PVA, to minimisenon-specific binding and to facilitate free movement of the labelledreagent after the macroporous body has become moistened with the liquidsample. The macroporous carrier material can be pre-treated with asurface active agent or solvent, if necessary, to render it morehydrophilic and to promote rapid uptake of the liquid sample. Suitablematerials for a macroporous carrier include plastics such aspolyethylene or glass-fibre. In the case that the labelled bindingreagent is labelled with a detectable particle, the macroporous body mayhave a pore size at least ten times greater than the maximum particlesize of the particle label. Larger pore sizes give better release of thelabelled reagent. As an alternative to a macroporous carrier, thelabelled binding reagent may be provided on a non-porous substrateprovided upstream from the detection zone, said non-porous substrateforming part of the flow-path.

The first and/or second assays may comprise a glass-fibre macroporouscarrier provided upstream from and overlapping at its distal end anitrocellulose porous carrier.

The assay device or kit may further comprise one or more opticaldetection means such as a photodetector and one or more light sourcessuch as an LED positioned so as to optically illuminate the detectionzones and determine the presence and/or amount of labelled speciespresent. The assay device may further comprise one or more of a powersource, a computation means, a signal transduction means, an algorithm,a display means, a memory means and data in/out port. The assay devicemay comprise a housing which serves to house the first and second assaysas well as other components of the device. The device may comprisestored threshold values.

The first and second assays may be provided for example in a side byside arrangement or in a face to face arrangement wherein one assay isprovided above the other. A sole optical means may be arranged to detectboth detection zones.

In addition to measuring the detection zones of the respective assays aswell as the control zones where present, the optical means may alsomeasure a reference zone, namely a portion of the flow-path which isfree from binding reagent in the dry state.

The purpose of the reference zone is to provide a signal value againstwhich the signal value obtained at the detection zone may be referenced.This takes into account any change in optical signal due to for example,the wetting of the porous carrier any differences in the opticalabsorption of the liquid sample.

An assay device for measuring the levels of analyte in a liquid samplecomprising an optical detection means arranged to measure the intensityof light reflected from a detection zone, control zone and referencezone of an assay device is described in EP1484601.

The illumination process may be carried out sequentially such thatdevice is able to know which from which zone light is being reflectedfrom onto the photodetector. The assay strips may be positioned in aside by side arrangement and the photodetector and light sourcespositioned above the plane of the strips such that the detection controland reference zones are positioned towards the light sources and opticaldetectors.

The first assay differs from the second assay such that the respectiveassays are capable of measuring analyte at different levels.

For example the first and second assays may employ differing assayarchitectures, such as the first assay employing a sandwich bindingreaction and the second assay employing a competition or inhibitionreaction. The first assay may comprise a mobilisable labelled bindingreagent for the analyte provided upstream from a detection zone, saiddetection zone comprising a non-labelled immobilised binding reagent forthe analyte and the second assay may comprise a mobilisable bindingreagent for the analyte provided upstream from an immobilisednon-labelled binding reagent for the mobilisable binding reagent.Alternatively the second assay may comprise a mobilisable labelledanalyte or analyte analogue reagent provided upstream from animmobilised non-labelled binding reagent for the analyte. The sandwichassay is the high sensitivity assay, namely it is capable of measuringanalyte at a lower concentration range and the inhibition or competitionassay is a low sensitivity assay, namely it is capable of measuringanalyte at a higher concentration range.

The assay device may for example comprise first and second assayswherein the non-labelled binding reagent of the first assay differ fromthe non-labelled binding reagent of the second assay, and/or thelabelled binding reagent of the first assay differs from the labelledbinding reagent of the second assay. For example this may be adifference in concentration, or a difference in affinity for an analyte,analyte analogue or binding reagent. A high affinity binding reagentwill have a higher analyte sensitivity than a lower affinity bindingreagent. Similarly a low concentration of binding reagent will have alower analyte sensitivity than a high concentration of binding reagent.The first and second assays may be varied in this way such that they arecapable of determining an analyte at different concentration ranges.

Thus the assay device may comprise a high analyte sensitivity firstassay comprising a mobilisable labelled binding reagent of a certainconcentration or affinity provided upstream from a detection zone and alow analyte sensitivity second assay comprising a mobilisable labelledbinding reagent having a lower concentration or affinity providedupstream from a detection zone. Alternatively or additionally, the firstassay comprising an immobilised binding reagent at a detection zone of acertain concentration or affinity and a second assay may comprise animmobilised binding reagent at a detection zone having a lowerconcentration or affinity.

Another way to lower the sensitivity of an assay is to reduce thebinding reagent to particle loading. Thus the assay device may comprisea first high analyte sensitivity assay and a second low analytesensitivity assay wherein the first assay comprises a mobilisableparticle labelled binding reagent provided upstream from a detectionzone having a ratio of binding reagent to particle label and wherein thesecond assay comprises a mobilisable particle labelled binding reagentprovided upstream from a detection zone having a lower ratio of bindingreagent to particle label than that of the first assay.

A further way to lower the sensitivity of an assay is to provide alabelled binding reagent having a label of a low optical density. Thismay be achieved for example by provision of a polymer particle labelhaving a low concentration of dye. Thus the assay device may comprise afirst high analyte sensitivity assay and a second low analytesensitivity assay wherein the first assay comprises a mobilisableparticle labelled binding reagent provided upstream from a detectionzone, said label having an optical density and wherein the second assaycomprises a mobilisable particle labelled binding reagent providedupstream from a detection zone wherein the label has a lower opticaldensity than that of the first assay.

Yet a further way to measure high analyte levels is to employ anon-particulate labelled binding reagent. High levels of analyte whenmeasured by way of a sandwich binding assay require high levels ofbinding reagent. In the case wherein the label is a particle label,provision of high levels of analyte within or on the porous carrier cangive rise to steric hindrance resulting in poor assay sensitivity.Conversely, at lower analyte levels, the use of a non-particle labelledbinding reagent can give rise to a low signal due to the low opticaldensity. However, at high analyte levels, non-particle labels may bepresent at sufficiently high levels to readily detected. Therefore theassay may comprise a first high analyte sensitivity assay comprising anoptically detectable particle labelled binding reagent provided upstreamfrom a detection zone and a second low analyte sensitivity assaycomprising an optically detectable non-particle labelled binding reagentprovided upstream from a detection zone. An example of a opticallydetectable non-particle label may be a dye. The dye may be fluorescent.

A yet further way to lower the sensitivity of the assay is to employ aporous carrier such as nitrocellulose having a higher flow rate. Thusthe assay device may comprise a first high analyte sensitivity assayhaving a porous carrier having a flow-rate and a second low analytesensitivity assay having a porous carrier having a higher flow rate thanthat of the first assay.

A further way to lower analyte sensitivity is to provide for a fastrelease of the labelled binding reagent from the porous carrier duringcontact with the liquid sample. Ways to increase the release of labelledbinding reagent are for example to increase the levels of sugars ormethylcellulose in the device.

A further way to lower analyte sensitivity is to employ a labelledbinding reagent having a lower optical sensitivity. One way of achievingthis to use a colour which is less sensitive to an optical detector.

Use of a Scavenger Reagent

A further way to lower the analyte sensitivity is to provide a scavengerbinding reagent to bind to analyte. The scavenger binding reagent may beprovided upstream from a detection zone and may be immobilised,mobilisable or both. The scavenger binding reagent may be provided ateither the same region of the porous carrier as the mobilisable bindingreagent, upstream from it or downstream from it. The scavenger bindingreagent may bind to the same binding region of the analyte as themobilisable labelled binding reagent or to a different region of theanalyte than the labelled binding reagent. Either or both of the assaysmay employ a scavenger binding reagent and the scavenger bindingreagents may differ from one another in terms of their concentration,affinity or both.

According to an embodiment, the assay device comprises a first assaycomprising a first porous carrier comprising a mobilisable labelledbinding reagent provided upstream from a detection zone and a secondassay comprising a mobilisable labelled binding reagent providedupstream from a detection zone and a scavenger binding reagent alsoprovided upstream from the detection zone of the second assay. The firstassay may be the high analyte sensitivity assay and the second assay maybe the low analyte sensitivity assay.

The scavenger reagent may be provided in a mobilisable form.

The scavenger reagent may have a different affinity for the analyte thanthe mobilisable labelled binding reagent of the second assay. In anexemplary embodiment, the scavenger binding reagent has a higheraffinity for the analyte than the mobilisable binding reagent of thesecond assay. The amount scavenger binding reagent may be varied tochange the sensitivity of the second assay to analyte concentration.Increasing the amount of scavenger binding reagent present lowers thesensitivity of the assay due to the fact that the scavenger bindingreagent is able to bind more analyte, effectively lowering theproportion of labelled binding reagent that is able to bind to thedetection zone. The amount of labelled binding reagent in the first andsecond assays may be varied. Increasing the amount of labelled bindingreagent has the tendency to reduce the hook effect and the amount oflabelled binding reagent present, especially in the lower sensitivityassay, may be varied depending upon the analyte range.

The scavenger binding reagent may be capable of binding to the same or adifferent analyte binding region. In an exemplary embodiment, thescavenger binding reagent is capable of binding to a different bindingregion of the analyte. In particular where the analyte to be determinedis hCG, the scavenger binding reagent is capable of binding to thebeta-subunit, and the mobilisable labelled binding reagent is capable ofbinding to the alpha-subunit.

According to an exemplary embodiment, the assay device comprises a firstassay comprising a glass-fibre porous carrier material comprising amobilisable particle-labelled binding reagent for an analyte and anitrocellulose porous carrier material provided downstream from theglass-fibre porous carrier material having a detection zone comprising aimmobilised non-labelled binding reagent for the analyte and a secondassay comprising a glass-fibre porous carrier material comprising amobilisable particle-labelled binding reagent for a first binding regionof the analyte and a mobilisable scavenger binding reagent for a secondbinding region of the analyte and a nitrocellulose porous carriermaterial provided downstream from the glass-fibre porous carriermaterial having a detection zone comprising an immobilised non-labelledbinding reagent for the second binding region of the analyte.

It will be appreciated that the above ways to alter the assaysensitivity of an assay are not be exhaustive. The assay device maycomprise one of more the above features to affect assay sensitivity. Theparticular assay architecture chosen would depend upon the analyte andits concentration range.

Other Embodiments

In addition to the embodiments, aspects and objects of the presentinvention disclosed herein, including the claims appended hereto, thefollowing paragraphs set forth additional, non-limiting embodiments andother aspects of the present invention.

In one aspect the invention provides a assay device for thedetermination of analyte in a liquid sample over an extendedconcentration range comprising a first assay and a second assay, whereinthe first assay for an analyte comprises a first flow-path having a soledetection zone capable of immobilising a labelled binding reagent andthe second assay for said analyte comprises a second flow-path having asole detection zone capable of immobilising a labelled binding reagent,wherein the presence of labelled binding reagent at the detection zonesprovides an indication of the presence and/or extent of analyte in saidliquid sample. The first assay is capable of providing an indication ofthe level of analyte in a first concentration range and the second assayis capable of providing an indication of the level of analyte in asecond concentration range. In certain embodiments the first and secondconcentration ranges overlap. The device is capable of providing anindication of the level of analyte with respect to one or morethresholds. In some embodiments, the first and/or second flow-pathcomprises a porous carrier, such as a lateral flow porous carrier.

In certain embodiments, the first and/or second assay comprises amobilisable labelled binding reagent for the analyte provided upstreamfrom a detection zone. In other embodiments, the detection zone of thefirst and/or second assay comprises an immobilised binding reagent forthe analyte. Optionally, the first assay defines a sandwich assay andthe second assay defines a competition or inhibition assay. In anotherembodiment, the first assay comprises a mobilisable labelled bindingreagent for the analyte provided upstream from a detection zone, saiddetection zone comprising an immobilised binding reagent for the analyteand wherein the second assay comprises a mobilisable binding reagent forthe analyte provided upstream from provided upstream from a detectionzone, said detection zone comprising an immobilised analyte or analyteanalogue for the mobilisable binding reagent. In other embodiments, thefirst assay comprises a mobilisable labelled binding reagent for theanalyte provided upstream from a detection zone, said detection zonecomprising an immobilised binding reagent for the analyte and whereinthe second assay comprises a mobilisable labelled analyte or analyteanalogue provided upstream from an immobilised binding reagent for theanalyte.

In certain embodiments, the first assay is a high analyte sensitivityassay and the second assay is a low analyte sensitivity assay. In otherembodiments, the first assay comprises a mobilisable labelled bindingreagent for an analyte provided upstream from a detection zone, whereinthe detection zone comprises an immobilised non-labelled binding reagentfor the analyte; and wherein the second assay comprises a mobilisablelabelled binding reagent for a first binding region of the analyte and ascavenger binding reagent for the analyte provided upstream from adetection zone, wherein said detection zone is capable of binding thelabelled binding reagent. For example, the detection zone of the firstand/or second assays comprises an immobilised binding reagent for theanalyte. In another embodiment, for the second assay, the scavengerbinding reagent has a higher affinity for the analyte than themobilisable labelled binding reagent. Similarly, the mobilisablelabelled binding reagent and the scavenger binding reagent of the secondassay are for respectively a first and second binding region of theanalyte, the detection zone comprising an immobilised binding reagentfor a second binding region of the analyte. In a related embodiment, themobilisable labelled binding reagent and the scavenger binding reagentof the second assay are provided in the same region. In another relatedembodiment, the scavenger binding reagent is provided in a mobilisableform. In certain embodiments, the mobilisable binding reagents and thescavenger reagent are provided on and/or in a first porous carriermaterial and the immobilised binding reagent provided at the detectionzone is provided on and/or in a second porous carrier material. Thefirst porous carrier material is, e.g., glass-fibre and the secondporous carrier material is nitrocellulose.

In some embodiments, the analyte is hCG. For example, the first bindingregion of hCG is the alpha sub-unit and the second binding region is thebeta sub-unit. Optionally, the binding reagents are antibodies, and/orthe binding reagent is labelled with an optically detectable particlesuch as a coloured polymer label. In certain embodiments, the fluidsample is urine.

In some embodiments, the devices of the invention include a commonsample application region which serves to supply liquid sample to bothassays. In other embodiments, the device includes a housing wherein thefirst and second assays are provided within said housing.

In another aspect, the invention provides an assay device fordetermining the presence and/or extent of an analyte in a liquid samplecomprising a first assay and a second assay wherein said first assaycomprises a porous carrier having a detection zone comprising animmobilised binding reagent for an analyte and provided upstream fromthe detection zone is a mobilisable labelled binding reagent for saidanalyte; and wherein said second assay comprises a detection zonecomprising an immobilised binding reagent for the analyte and providedupstream from the detection zone is a mobilisable labelled bindingreagent for the analyte and a scavenger reagent for said analyte,wherein detection of the presence of labelled binding reagent at thedetection zone provides an indication of the presence and/or extent ofanalyte in said liquid sample. In some embodiments, the first assay is ahigh sensitivity assay and the second assay is a low analyte sensitivityassay. In other embodiments, the analyte is hCG.

The invention provides devices in which the first assay either providesan indication of whether the subject is pregnant or not, based uponwhether the level of hCG detected is respectively above or below a basethreshold, and/or if pregnant, the level of hCG in a first range of lessthan or equal to a first threshold, the second assay provides anindication of the level of hCG in a second range of above or equal to asecond threshold and the first and second assays together provide anindication of the level of hCG in a third range of greater than thefirst threshold but less than the second threshold. The invention alsoprovides devices in which for the second assay, the scavenger bindingreagent has a higher affinity for the analyte than the mobilisablelabelled binding reagent.

In a further aspect, the invention provides an assay kit for determiningthe presence an/or extent of an analyte in a liquid sample comprising afirst assay and a second assay wherein said first assay comprises aporous carrier having a detection zone comprising an immobilised bindingreagent for an analyte and provided upstream from the detection zone isa mobilisable labelled binding reagent for said analyte; and whereinsaid second assay comprises a detection zone comprising an immobilisedbinding reagent for the analyte and provided upstream from the detectionzone is a mobilisable labelled binding reagent for the analyte and ascavenger reagent for said analyte, wherein detection of the presence oflabelled binding reagent at the detection zones provides an indicationof the presence and/or extent of the analyte in the sample.

In another aspect, the invention provides a method of fabricating anassay device to reduce a hook effect, wherein the assay device comprisesa first flow-path and a second flow-path, comprising the steps ofproviding a first flow-path comprising a porous carrier and a secondflow-path comprising a porous carrier; contacting the first flow-pathwith a first material so as to form a first zone; and contacting thesecond flow-path with a first material so as to form a second zone,wherein the first zone and the second zone are separated by a distanceof at least about 5 mm, and whereby the hook effect is thereby reduced.In certain embodiments, the first material contains latex.

Comparative Example 1 Preparation of an Assay Device Comprising a SinglePorous Carrier Comprising a First Upstream Detection Zone for a SandwichAssay and a Second Downstream Detection Zone for an Inhibition Assay

An assay test-strip comprising a first upstream detection zone for asandwich assay and the second downstream detection zone for aninhibition assay and a mobilisable labelled binding reagent providedupstream from said zones was prepared as follows:

A solution of 1.5 mg/ml mAb mouse anti-human β-hCG (clone 3468, suppliedin house) in PBSA buffer and 7.2 KIU/ml hCG (Scipac) in PBSA/ovalbuminwas mixed for 1-hour to provide an anti-β hCG-hCG conjugate. Theresulting solution was plotted in the form of a line onto nitrocellulosestrips (Whatman) of dimensions 40 mm length×6 mm width, having apore-size of 8 microns and a thickness between 90-100 microns which hadbeen laminated to a 175 micron backing layer. The conjugate wasdispensed at a concentration of 1.5 mg/ml and a rate of 1 μl/cm at the16 mm position (distance along the nitrocellulose) using a Biodotxyz3050 dispensing platform to form a second downstream detection zone.

The first detection zone was prepared by plotting a line of anti-humanβ-hCG antibody (clone 3468) at a concentration of 3 mg/ml in PBSA bufferand a rate of 1 μl/cm on the nitrocellulose at the 10 mm position usingthe Biodot xyz3050 dispensing platform. The nitrocellulose was heatedbriefly to 55° C. and blocked with a solution of 1% w/w PVA in 0.05% w/wTween-20 in pH9Tris-buffered saline/5% ethanol, followed by treatmentwith 2% w/w sucrose in de-ionised water. The nitrocellulose wasthereafter heated briefly at 75° C.

Mouse-anti-human α-hCG mAb (clone 3299, supplied in-house) wasconjugated to 400 nm blue latex polystyrene latex (Duke Scientific) inBSA/sucrose and sprayed onto a glass-fibre pad (F529-09, Whatman) at arate of 50 g/hr and 110 mm/s. The glass fibre pad was partially overlaidonto and upstream from the nitrocellulose porous carrier.

Example 1

Assay devices were prepared in a similar manner to that of ComparativeExample 1 except that the first and second detection zones were providedon respectively first and second test-strips, wherein the first andsecond detection zones were provided on nitrocellulose, each test-stripcomprising glass-fibre sprayed with mobilisable latex labelled α-hCGantibody provided upstream from the nitrocellulose.

Preparation of the First Test-Strip

The detection zone was prepared by plotting a line of anti-human β-hCGantibody (clone 3468) at a concentration of 3 mg/ml in PBSA buffer and arate of 1 μl/cm on the nitrocellulose at the 10 mm position using theBiodot xyz3050 dispensing platform. The nitrocellulose was heatedbriefly to 55° C. and blocked with a solution of 1% w/w PVA in 0.05% w/wTween-20 in pH9Tris-buffered saline/5% ethanol, followed by treatmentwith 2% w/w sucrose in de-ionised water. The nitrocellulose wasthereafter heated briefly at 75° C.

Mouse-anti-human α-hCG mAb (clone 3299, supplied in-house) wasconjugated to 400 nm blue latex polystyrene latex (Duke Scientific) inBSA/sucrose and sprayed onto a glass-fibre pad (F529-09, Whatman) at arate of 50 g/hr and 110 mm/s. The glass fibre pad was partially overlaidonto and upstream from the nitrocellulose porous carrier.

Preparation of Second Test-Strip

A solution of 1.5 mg/ml mAb mouse anti-human β-hCG (clone 3468, suppliedin house) in PBSA buffer and 7.2 KIU/ml hCG (Scipac) in PBSA/ovalbuminwas mixed for 1-hour to provide an anti-β hCG-hCG conjugate. Theresulting solution was plotted in the form of a line onto nitrocellulosestrips (Whatman) of dimensions 40 mm length×6 mm width, having apore-size of 8 microns and a thickness between 90-100 microns which hadbeen laminated to a 175 micron backing layer. The conjugate wasdispensed at a concentration of 1.5 mg/ml and a rate of 1 μl/cm at the16 mm position (distance along the nitrocellulose) using a Biodotxyz3050 dispensing platform to form the detection zone.

Mouse-anti-human a-hCG mAb (clone 3299, supplied in-house) wasconjugated to 400 nm blue latex polystyrene latex (Duke Scientific) inBSA/sucrose and sprayed onto a glass-fibre pad (F529-09, Whatman) at arate of 50 g/hr and 110 mm/s. The glass fibre pad was partially overlaidonto and upstream from the nitrocellulose porous carrier.

The test-strips according to Example 1 and Comparative Example 1 weretested using in-house readers with calibrated hCG buffer standards atconcentrations 0, 25 50, 100, 250, 500, 1000, 2500, 5000, 10000, 15000,20000, 25000, 50000, 150000, 200000 and 250000 mIU/ml hCG.

The signal intensity measured at the inhibition detection zones as afunction of hCG concentration of the assays of example 1 (denoted by--♦--) and comparative example 1 (denoted by --▪--) is shown in FIG. 2as signal in arbitrary units vs. mIU/ml hCG.

As can be seen from this Figure, the inhibition detection zone ofComparative Example 1 shows an initial plateau at levels of hCG rangingfrom 0-100 mIU/ml, followed by a decrease in the intensity at higherlevels of hCG as expected. However, at higher levels still, the signalintensity was observed to increase. By comparison, the signal intensityof Example 1 decreases at higher hCG levels without the subsequentincrease in signal intensity at higher hCG levels. As can be seen, theinhibition zone of assay device constructed according to ComparativeExample 1 has a more limited range over which hCG may be measured.

Example 2 Preparation of Assay Devices Comprising a First Test-StripComprising a First Sandwich Assay and a Second Test-Strip Comprising aScavenger Assay in Addition to a Sandwich Assay Preparation of theSecond Test-Strip.

MAb mouse anti-human β-hCG antibody (clone 3468) at a concentration of 3mg/ml in PBSA buffer was plotted onto nitrocellulose (of type anddimensions as that according to Comparative Example 1) at the 10 mmposition at a rate of 1 μl/cm using a Biodot XYZ3050 dispensing platformto provide a sole detection zone for the first assay.

Mouse-anti-human α-hCG mAb (clone 3299) conjugated to 400 nm bluepolystyrene latex (Duke Scientific) was mixed with scavenger antibodymAb mouse anti-human β-hCG (clone 3468) at 3 mg/ml to give a final %blue latex of 3% and a final 3468 concentration of 0.075 mg/ml. Theresulting mixture was sprayed at 2.02 μg/cm onto F529-09 glass fibre.

The glass fibre was partially overlaid and provided upstream from thenitrocellulose to provide the first assay test-strip.

Preparation of the First Assay Test-Strip

The first assay test strip was prepared according to the first assaytest-strip according to that of Example 1.

Comparative Example 2

Assay devices constructed wherein both detection zones were provided onthe same porous carrier were not able to result in the measurement of ananalyte concentration over an extended analyte range.

Assay devices according to Example 2 were tested using in-housedetection zone optical readers with calibrated hCG buffer standards at12 concentrations ranging from 0-250000 mIU/ml hCG. 10 replicates perconcentration level were measured giving a total number of assay devicesthat were tested of 120.

The signal intensity vs hCG concentration for the second assayconstructed according to Example 2 is shown in FIG. 3.

The first assay test-strip according to Example 2 was able to determinethe amount of hCG present up to about 400 mIU/ml before the assay curveflattened off. The second assay test-strip according to Example 2 wasable to detect hCG levels of greater than about 1000 mIU/ml. Measurementof the signals at both the first and second assay test-strips enableddetermination of the level of hCG between about 400 mIU/ml and 1000mIU/ml.

The Effect of Varying the Amounts of Scavenger Antibody

Second assay test-strips according to Example 2 were prepared except theamount of scavenger antibody present was varied during preparation ofthe strip to give a final 3468 concentration of 0.12, 0.16, 0.2 and 0.24mg/ml.

As can be seen from FIG. 4 increasing the amount of scavenger antibodylowers the amount of analyte captured at the detection zone.

Example 3

The aim of this Example is to assess the effect of changing secondarylatex spray position on assay curve performance, with the intent ofreducing the hook effect. Materials used are as follows:

Latex 2 Reagent Composition Latex 1 (Secondary latex) Test latex 3%solids, anti β hCG 42.25 mls 42.25 mls Control latex 0.7% solids, Goatanti none 16.83 mls rabbit IgG Free antibody anti β hCG  1.16 mls  1.16mls

Two latex sprays were generated. For Latex spray 1, the distance frombase of glass fibre was held constant at 5 mm, while for Latex spray 2,the distance from base of glass fibre was set at 7, 9, 10, 11, 13, 14,or 16 mm.

As shown below, the signal was measured at 3 minutes run time as apercentage attenuation (% A) of the test line.

Distance from Base [hCG] mIU/ml of Glass Fibre (mm) 400 1000 2000 1000050000 250000 7 17.74 29.66 39.44 49.80 48.15 30.10 9 14.49 22.54 31.0754.90 50.98 34.23 10 15.63 23.11 32.76 59.34 61.04 42.57 11 16.68 24.6233.11 58.65 61.47 46.54 13 13.88 20.71 29.33 57.66 62.56 47.99 14 17.3522.82 31.87 58.40 64.42 48.82 16 14.69 19.86 28.75 56.55 63.81 49.20

Results shown above and in FIG. 5 demonstrate that if secondary latexspray position is below 9 mm, the % A signal at the higher end of thecurve falls greatly (for example, signal at 250,000 mIU/ml hCG dropsbelow signal 2000 mIU/ml). It can be concluded that increasing latexspray position to 10 mm or greater, e.g., 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30 or greater than 30, significantlyreduces the hook effect.

1. An assay device for determining the presence or concentration of ananalyte in a liquid sample over an extended concentration rangecomprising: a) a first flow-path having a sole detection zone capable ofimmobilising a labelled binding reagent, the first flow-path capable ofproviding an indication of the level of analyte in a first concentrationrange; and b) a second flow-path having a sole detection zone capable ofimmobilising a labelled binding reagent, the second flow-path capable ofproviding an indication of the level of analyte in a secondconcentration range; wherein the presence of labelled binding reagent atthe detection zones provides an indication of the presence orconcentration of analyte in said liquid sample.
 2. (canceled)
 3. Thedevice according to claim 1 wherein the first and second concentrationranges overlap.
 4. The assay device according to claim 1 which iscapable of providing an indication of the level of analyte with respectto one or more thresholds.
 5. The device according to claim 1 whereinthe first and/or second flow-path comprises a porous carrier. 6.(canceled)
 7. The device according to claim 1 wherein the first and/orsecond flow-path comprises a mobilisable labelled binding reagent forthe analyte provided upstream from a detection zone.
 8. The deviceaccording to claim 1 wherein the detection zone of the first and/orsecond flow-path comprises an immobilised binding reagent for theanalyte.
 9. The device according to claim 1 wherein the first flow-pathdefines a sandwich assay and the second flow-path defines a competitionor inhibition assay.
 10. The device according to claim 9 wherein thefirst flow-path comprises a mobilisable labelled binding reagent for theanalyte provided upstream from a detection zone, said detection zonecomprising an immobilised binding reagent for the analyte and whereinthe second flow-path comprises a mobilisable binding reagent for theanalyte provided upstream from provided upstream from a detection zone,said detection zone comprising an immobilised analyte or analyteanalogue for the mobilisable binding reagent.
 11. The device accordingto claim 9 wherein the first flow-path comprises a mobilisable labelledbinding reagent for the analyte provided upstream from a detection zone,said detection zone comprising an immobilised binding reagent for theanalyte and wherein the second flow-path comprises a mobilisablelabelled analyte or analyte analogue provided upstream from animmobilised binding reagent for the analyte.
 12. (canceled)
 13. Thedevice according to claim 1 wherein the first flow-path comprises amobilisable labelled binding reagent for an analyte provided upstreamfrom a detection zone, wherein the detection zone comprises animmobilised non-labelled binding reagent for the analyte; and whereinthe second assay comprises a mobilisable labelled binding reagent for afirst binding region of the analyte and a scavenger binding reagent forthe analyte provided upstream from a detection zone, wherein saiddetection zone is capable of binding the labelled binding reagent. 14.(canceled)
 15. (canceled)
 16. The device according to claim 13 whereinthe mobilisable labelled binding reagent and the scavenger bindingreagent of the second flow-path are for respectively a first and secondbinding region of the analyte, the detection zone comprising animmobilised binding reagent for a second binding region of the analyte.17. (canceled)
 18. (canceled)
 19. The device according to claim 13wherein the mobilisable binding reagents and the scavenger reagent areprovided on or in a first porous carrier material and the immobilisedbinding reagent provided at the detection zone is provided on and/or ina second porous carrier material.
 20. The device according to claim 19wherein the first porous carrier material is glass-fibre and the secondporous carrier material is nitrocellulose.
 21. (canceled)
 22. (canceled)23. (canceled)
 24. The device according to claim 1 wherein the fluidsample is urine.
 25. The device according to claim 1 wherein the bindingreagent is labelled with an optically detectable particle.
 26. Thedevice according to claim 25 wherein the optically detectable particleis a coloured polymer label.
 27. The device according to claim 1,comprising a common sample application region which serves to supplyliquid sample to both flow-paths.
 28. (canceled)
 29. An assay device fordetermining the presence or concentration of an analyte in a liquidsample comprising a first flow-path and a second flow-path wherein saidfirst flow-path comprises a porous carrier having a detection zonecomprising an immobilised binding reagent for an analyte and providedupstream from the detection zone is a mobilisable labelled bindingreagent for said analyte; and wherein said second flow-path comprises adetection zone comprising an immobilised binding reagent for the analyteand a mobilisable labelled binding reagent upstream from the detectionzone for the analyte and a scavenger reagent for said analyte, whereindetection of the presence of labelled binding reagent at the detectionzone provides an indication of the presence or concentration of analytein said liquid sample.
 30. (canceled)
 31. (canceled)
 32. The deviceaccording to claim 1, wherein: a) the first flow-path is capable ofproviding an indication of the level of hCG in the sample in a firstrange of less than or equal to a first threshold; and b) the secondflow-path is capable of providing an indication of the level of hCG inthe sample in a second range of above or equal to a second threshold;and c) the first and second flow-paths together are capable of providingan indication of the level of hCG in a third range of greater than thefirst threshold but less than the second threshold.
 33. (canceled) 34.(canceled)
 35. An assay kit for determining the presence orconcentration of an analyte in a liquid sample comprising an assaydevice comprising a first flow-path and a second flow-path wherein: a)said first flow-path comprises: i) a porous carrier having a detectionzone comprising an immobilised binding reagent for an analyte; and ii) amobilisable labelled binding reagent for said analyte upstream from thedetection zone; and b) said second flow-path comprises: i) a detectionzone comprising an immobilised binding reagent for the analyte; and ii)a mobilisable labelled binding reagent for the analyte and a scavengerreagent for said analyte upstream from the detection zone, whereindetection of the presence of labelled binding reagent at the detectionzones provides an indication of the presence and/or extent of theanalyte in the sample. 36-38. (canceled)